Bacteria-mediated diarrhoeal diseases in humans include cholera, typhoid fever, traveller's diarrhoeal and acute diarrhoeal illness in infants (Black, 1993, Vaccine 11: 100-106). Typhoid fever is representative of the invasive type which is characterized by invasion of the intestinal mucosa by the pathogen. In the non-invasive type, the symptoms are the result of a bacterial toxin which stimulates an enormous increase in the secretory activity of the cells lining the small intestine causing an acute loss of body fluid. An example of the non-invasive type of pathogen is enterotoxigenic E. coli (ETEC), which causes diarrhoea, particularly in children of developing countries and travellers visiting such areas. In fact, this pathogen has recently been identified by the World Health Organization (WHO) as one of the targeted pathogens for control by vaccination (Sansonetti, 1998 Nature Med 4: 499-500). In addition to diarrhoeal disease, pathogenic strains of E. coli cause urinary tract infections and hamburger disease: urinary tract infections caused by E. coli send 1.5 million people to the hospital each year in the United States alone and seven million more to their doctors (Service, 1997 Science 276: 533) and the recent outbreaks of hamburger disease in humans caused by E. coli 0157:H7 strain (MMWR Morb. Mortal. Wkly. Rep., 1999) are reminders of the ability of bacteria to rapidly adapt to new environmental conditions and new antibiotic counter-measures.
It has also been shown that ETEC-mediated diarrhoeal diseases also affect agricultural animals, especially cattle and pigs. Because calves and piglets are particularly susceptible, the agricultural industry suffers a sizeable loss of livestock each year from outbreaks of these diseases. In the case of piglets, pre-weaning diarrhea occurs shortly after birth from 1 to 7 days of age. In one of the surveys of pre-weaning disease in pigs, diarrhoea had the highest morbidity and represented 11% of the pre-weaning mortality with ETEC being the primary and sole infectious cause (Alexander, 1994 in: Gyles (ed), Escherichia coli in domestic animals and humans. (CAB International: Wallingford, UK.), pp. 151-170). A second disease, referred to as post-weaning ETEC diarrhoea typically starts 3 to 10 days after weaning. Otherwise, post-weaning diarrhea occurs after weaning in 22 to 49 day old piglets. This disease is a major cause of economic losses to swine industry from both mortality and reduced growth rates and is the most common cause of post-weaning mortality in many farms, killing 1.5 to 2% of pigs weaned (Hampson, 1994 in: Gyles (ed), Escherichia coli in domestic animals and humans. (CAB International: Wallingford, UK), pp. 171-191).
The strains of ETEC that are associated with intestinal colonization are those that express the K88, K99, 987P, F41 and F18 fimbrial adhesions and enterotoxins (Sta, Stb, LT and Vte) (Ojeniyi et al., 1994, J. Vet Med. 41: 49-59; Imberechts et al., 1997, Vet Microbiol 54: 329-341). These adhesions are located in the rod-like pili (fimbriae) that extend from the E. coli and are bound to specific receptors on the intestinal wall. Among the different ETEC, those expressing the K88 fimbrial antigen are the most prevalent form of E. coli infection, being found world-wide wherever pigs are raised in high numbers (Rapacz and Hasler-Rapacz, 1986, Animal Gen 17: 305-321). Specifically, it has been estimated that K88 ETEC is responsible for 50% of the 10 million piglet deaths each year (Waters and Sellwood, 1982 in: Proceedings of the 2nd World Congress on Genetics Applied to Livestock Production, Madrid, Spain, pp. 362). There are significant concerns that diarrhoea in neonatal and post-weaning pigs will become more serious in the future, given the trend towards large herds, early weaning, increased incidence of antibiotic resistance in microorganisms and pressure by regulatory agencies to ban or reduce the use of antibiotics in feeds. In addition, there is strong evidence to suggest that resistance in an animal pathogen can readily be transmitted to a human pathogen. Clearly, one pathogen that is able to mutate rapidly is E. coli, as evidenced by the many antibiotic resistant strains that have appeared. As such, the possibility exists that resistance in livestock pathogens could be transferred to human pathogens. Thus, there is a need to develop alternate strategies to control this organism in human and veterinary medicine, particularly in swine industry.
One of the alternative strategies for controlling the pathogens is vaccination. Traditional vaccines fall into two broad categories: attenuated, non-pathogenic live infectious material; and killed, inactivated, or subunit preparations. Although live attenuated vaccines produce a diverse and persistent immune response, they present safety concerns due to the risk of reversion during replication or mutation to become infectious. Although non-live vaccines do not induce infection, the immunity induced by such vaccines can frequently wane during the life span of inoculated hosts and often requires repeat boosting to achieve lifelong immunity.
Thus to control diarrhoeal diseases in animals and humans, vaccination against specific strains of ETEC using vaccines based on live attenuated or killed organism or recombinant peptide or protein subunit could be an alternative strategy. Use of vaccines based on immunogenic material of E. coli, selected from pili, pili proteins and E. coli antigens for controlling diarrhoeal disease in livestock has been described by Lutticken, et al., (U.S. Pat. No. 4,788,056). The most important ETEC virulence associated factors are toxins and colonization factor antigens (CFAs), which are usually fimbrial adhesions on the bacterial surface (Svennerholm et al, 1989, Vaccine 7: 196-198). There are several reports on the preparation of genetic vaccines based on colonization factor antigens (CFAs) of ETEC (Alves et al., 1998, Vaccine 16: 9-15; Alves et al., 1999, Brazilian J Med Biol Res 32: 223-229, Alves et al., 1999, FEMS Immunol Med Microbiol 23: 321-330). Specifically, Alves discloses a DNA vaccine for direct vaccination of mice. The vaccine comprises CFA/I fused to glycoprotein D of HSV type 1 virus under the control of Rous Sarcoma Virus promoter. Maas, et al. in U.S. Pat. No. 4,761,372 described the preparation of DNA plasmids containing genes coding for non-toxic heat-labile enterotoxin, non-toxic heat-stable enterotoxin and colonization factor. The plasmids were used to transform E. coli and the bacteria containing the plasmids were used as a vaccine.
It is known that the vaccination of a sow against specific strains of E. coli results in the secretion of colostrum that will provide passive immunity to the piglets against ingested ETEC. This protection, however, is transient and all protection is lost shortly after weaning. Furthermore, immunization of the piglet is also not practical as colibacillosis will often develop in the piglet before the piglet develops immunity to the inoculum, meaning that direct immunization will not protect such pigs against pathogenic ETEC (Alexander, 1994; Hampson, 1994; Isaacson, 1994 in: Gyles, G. L. (ed), Escherichia coli in domestic animals and humans (CAB International: Wallingford, UK), pp. 629-647).
The fimbrial antigens of porcine ETEC that are associated with intestinal colonization have been extensively investigated with respect to their genetic background, protein chemistry, and immunological properties. As discussed above, purified fimbrial antigens (Husband and Serman, 1979, Austral. Vet. J. 55: 435436) and recombinant proteins (Isaacson, 1985, Avian Diseases 30: 28-36; Gyles and Mass, 1987, Prog. Vet Microbiol. Immun. 3: 139-158) have been widely used with promising results as vaccine antigens in controlling colibacillosis in pigs (Isaacson, 1994). In passive immunization experiments, chicken egg-yolk antibodies raised against the different fimbrial antigen when administered orally, have also been shown to be highly effective at protecting neonatal pigs against fatal enteric colibacillosis (Yokoyama et al., 1992, Infect Immun. 60: 998-1007; Marquardt et al., 1999, FEMS Immun. Med. Microbiol. 23: 283-288). The disadvantages of using protein immunization is that the protein must be purified and injected frequently in the presence of an adjuvant if a high and sustained antibody titre is to be obtained. This is an expensive, time consuming and invasive procedure. In addition, isolation of antigens from human pathogens is potentially dangerous.
One highly attractive and effective alternative approach for the control of pathogens, including E. coli, is to use therapeutic antibodies. These antibodies can be produced in any animal and can be administered orally to another animal to control a specific disease. The advantage of using antibodies is that they provide a long term and sustainable means of controlling pathogens. Such a treatment would be highly effective, would not result in the development of resistant strains of pathogens, would spare the use of antibiotics and could be relatively inexpensive. Antibodies can be obtained from several sources including the colostrum of lactating animal, blood of animals, transgenic animals, transgenic plants, hybridoma cell lines, recombinant microorganisms and chicken eggs. Unfortunately, use of antibodies from the colostrum of lactating animals, particularly dairy cattle, is impractical as the colostrum is only produced over a short period of time. Currently, there is no information on the ability of antibodies from spray-dried plasma to counteract different intestinal pathogens and current methods for monoclonal antibody production either in hybridoma cell lines or transgenic animals/plants or recombinant microorganisms are prohibitively expensive.
The chicken egg (especially, yolk of the egg) is recognized as a rich source of specific antibodies (Gassmann, et al., 1990, FASEB J 4: 2528-2538; Tokoro, 1992 in U.S. Pat. No. 5,080,895). Tokoro described a method for treating an intestinal infectious disease in a neonatal mammal caused by a pathogenic organism with antibody-containing substance being obtained from the whole egg or albumen of the egg or the yolk of eggs laid by hens which have been immunized using a pathogenic organism as an antigen. This method claimed to be useful in treating diarrhoea in a neonatal mammal caused by ETEC. This suggests the potential of producing more specific antibodies against mammalian antigens in chickens compared to mammals because of the phylogenic distance between birds and mammals, low cost of production and convenience, and more importantly, the compliance with regulations for modern livestock production. Specifically, chickens produce eggs non-invasively and due to the phylogenic distance, the adjuvant does not cause severe responses as it does in mammals. It has also been shown that the production and maintenance of high levels of specific antibodies over a long period of time are possible in laying hens. Also, it is now possible to obtain antigen specific antibodies from egg-yolk of hyper-immunized hens. A hen lays 200-300 eggs per year and one egg yolk contains about 150 mg of antibodies. The yolk or the purified antibody can be dried by freeze-drying or spray-drying without the loss of activity and can be fed directly to the young pigs to provide protection against specific ETEC (Marquardt, et al., 1994 in: New Emerging Ova-Biotechnology (Ed. Sim and Nakai) CAB International: Wallingford, UK). When antibodies raised against ETEC (i.e. K88) fimbrial antigens were administered orally to neonatal and early-weaned piglets, they were protected against ETEC infections (Marquardt, et al., 1999 FEMS Immunol Med Microbiol 23: 283-288). It is of note that purified or partially purified peptides were used to elicit the immune response in the host, which served as the source for the antibodies. However, there are certain disadvantages in using fimbrial proteins as antigens for producing antibodies in chicken eggs. Firstly, the specific antibody titre obtained in the egg yolk is only moderate compared to that obtained by using larger proteins as antigens. Secondly, the antigen must be administered to chickens several times in a year of laying cycle so as to maintain high antibody titre. Therefore, the production of antibodies using fimbriae as antigens is relatively expensive.
Clearly, a more effective, less costly method of producing anti-ETEC antibodies in chickens for use in passive immunization is needed.